Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, The National Institutes of Health, Bethesda, MD, 20892, USA.
Laboratory of Immunogenetics Twinbrook Imaging Facility, National Institute of Allergy and Infectious Diseases, The National Institutes of Health, Rockville, MD, 20852, USA.
BMC Biol. 2019 Dec 2;17(1):97. doi: 10.1186/s12915-019-0714-9.
Cellular functions can be regulated by cell-cell interactions that are influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources, but, as nutrients become depleted, they initiate a multi-cell developmental program that is dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor.
We show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth.
We have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development and to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression are enriched at centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell-autonomous functions for regulation of cellular adhesion and growth.
细胞功能可通过细胞间相互作用进行调节，而细胞外的密度依赖信号因子会影响这种调节。在富含营养物质的来源中，粘菌以单个细胞的形式生长，但随着营养物质的消耗，它们会启动一个多细胞发育程序，这个程序依赖于细胞密度的阈值。我们假设新的分泌蛋白可能作为密度感应因子，在特定的细胞密度阈值下促进多细胞发育命运的决定，并利用粘菌来鉴定这种因子。
我们发现，粘菌的多细胞发育聚集在局部细胞密度降低 2 倍时就会丢失。值得注意的是，在非允许的细胞密度下，发育聚集反应可以通过添加来自高密度、发育能力强的细胞的条件培养基来挽救。我们使用低细胞密度下细胞的聚集恢复作为测定方法，纯化了一种具有密度聚集活性的 150kDa 细胞外蛋白。MS/MS 肽序列分析确定了基因序列，并且过表达全长蛋白的细胞积累了更高水平的促进发育的因子（DPF）活性，从而使细胞在更低的细胞密度下聚集；缺乏这种 DPF 基因的细胞缺乏密度依赖性发育聚集活性，并且与 WT 相比，需要更高的细胞密度才能聚集。密度聚集活性与 DPF 的标记版本共纯化，并且标签亲和纯化的 DPF 具有密度聚集活性。在与 WT 的混合发育中，过表达 DPF 的细胞优先定位于多细胞聚集的中心，并且在细胞分化过程中定义细胞命运选择。最后，我们发现 DPF 作为一种较大的前体、单次跨膜蛋白合成，p150 片段通过蛋白水解切割和外显子脱落释放。DPF 的 TM/细胞质结构域具有细胞自主的细胞-基质粘附活性和细胞生长活性。
我们已经纯化了一种新的分泌蛋白 DPF，它作为发育的密度感应因子发挥作用，定义了粘菌发育的局部集体阈值，并促进细胞间通讯和多细胞形成。DPF 高表达区域在细胞-细胞信号反应、多细胞形成和细胞命运决定的中心富集。此外，DPF 具有调节细胞粘附和生长的独立的细胞自主功能。
Zotero 插件
